The semaphorins are a large group of extracellular proteins involved in a variety of processes during development, including neuronal migration and axon guidance. They function as chemorepellents that direct axons away from tissues marked by their expression, but in some cases can also act as chemoattractants. The semaphorin receptors are multiprotein complexes, which include a plexin molecule serving as the signal-transducing subunit. In addition, some semaphorin receptors include a neuropilin ligand-binding subunit. Research in this proposal focuses on detailed structural characterization of the semaphorins, neuropilins, plexins, and their interactions. Preliminary studies of semaphorins and neuropilins have identified their interacting domains. The crystal structure of the receptor-binding module of Semaphorin-3A was determined at 2.8 A resolution, revealing an unexpected beta propeller molecular architecture. X-ray crystallography will be used next to determine the structure of full-length Semaphorin-3A and structures of selected semaphorins from other classes. The analysis of these structures will focus on identifying structural elements important for defining the receptor and co-receptor specificities of semaphorins. The crystal structures of the interacting domains of neuropilins and plexins will also be determined. Finally, the structures of a series of semaphorin/neuropilin, neuropilin/plexin, semaphorin/plexin, and semaphorin/neuropilin/plexin complexes will be determined, illuminating the molecular events leading to the initiation of semaphorin signaling. The structural data will be complemented by biophysical, biochemical and flourescence/FRET-based studies of the semaphorin/neuropilin/plexin interactions. The combined structural and biophysical information will provide fundamental insights into the molecular mechanisms underlying the biological functions of semaphorins, neuropilins, and plexin, and could have significant applications in development of therapeutic agents for treatment of brain and spinal cord injuries.